Matrix Metalloproteinases Are Modifiers of Huntingtin Proteolysis

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Neuron Article

Matrix Metalloproteinases Are Modiﬁers of Huntingtin Proteolysis and Toxicity in Huntington’s Disease

John P. Miller,1 Jennifer Holcomb,1 Ismael Al-Ramahi,2 Maria de Haro,2 Juliette Gafni,1 Ningzhe Zhang,1 Eugene Kim,2 Mario Sanhueza,2 Cameron Torcassi,1 Seung Kwak,3 Juan Botas,2 Robert E. Hughes,1,* and Lisa M. Ellerby1,* 1Buck Institute for Age Research, Novato, CA 94945, USA 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 3CHDI Foundation, Inc., 300 Alexander Park, Suite 110, Princeton, NJ 08540, USA *Correspondence: [email protected] (R.E.H.), [email protected] (L.M.E.) DOI 10.1016/j.neuron.2010.06.021

SUMMARY Wellington et al., 2002). Inhibition of mutant Htt cleavage reduces and in some cases prevents toxicity in vitro and in vivo, indicating Proteolytic cleavage of huntingtin (Htt) is known to be a causative role for Htt proteolysis in HD pathogenesis (Gafni et al., a key event in the pathogenesis of Huntington’s 2004; Graham et al., 2006; Kim et al., 1999; Wellington et al., 2000). disease (HD). Our understanding of proteolytic A number of proteases have been shown to cleave mutant Htt. processing of Htt has thus far focused on the protease Caspases and calpains are the most widely studied and cleave families—caspases and calpains. Identifying critical Htt within the N-terminal region between amino acids 469 and proteases involved in Htt proteolysis and toxicity 586 (Gafni et al., 2004; Wellington et al., 2000). Recent work has identified Htt cleavage sites closer to the N terminus, using an unbiased approach has not been reported. between amino acids 105 and 167 (Lunkes et al., 2002; To accomplish this, we designed a high-throughput Ratovitski et al., 2007; Tanaka et al., 2006), producing smaller western blot-based screen to examine the generation and potentially more toxic mutant polyQ-containing fragments. of the smallest N-terminal polyglutamine-containing While these studies have suggested that cathepsins and/or Htt fragment. We screened 514 siRNAs targeting the calpains are the proteases responsible for producing these repertoire of human protease genes. This screen smaller fragments, the exact cleavage sites and identity of the identified 11 proteases that, when inhibited, reduced proteases involved have not been unequivocally identified Htt fragment accumulation. Three of these belonged (Kim et al., 2006; Lunkes et al., 2002; Ratovitski et al., 2007). to the matrix metalloproteinase (MMP) family. One Historically, Htt was the first neurodegenerative disease family member, MMP-10, directly cleaves Htt and protein identified as a caspase substrate, and our understanding prevents cell death when knocked down in striatal of proteolytic processing of Htt has thus far focused on the cysteine protease families of caspases and calpains (Gafni Hdh111Q/111Q cells. Correspondingly, MMPs are acti- et al., 2004; Goldberg et al., 1996; Graham et al., 2006; Hermel vated in HD mouse models, and loss of function of et al., 2004; Wellington et al., 1998, 2002; Yanai et al., 2006). In Drosophila homologs of MMPs suppresses Htt- this study, we used an unbiased approach to identify other induced neuronal dysfunction in vivo. protease families involved in Htt proteolysis and toxicity. From a drug development perspective, this is a particularly attractive approach given that there are 514 known and predicted protease INTRODUCTION genes present in the human genome, and many of these have existing inhibitory compounds developed as therapeutics for Huntington’s disease (HD) is a dominantly inherited neurodegen- a number of human diseases. One such class of proteases, the erative disorder that primarily affects neurons in the striatum and matrix metalloproteinases (MMPs) is involved in a number of cortex (Albin, 1995; Cudkowicz and Kowall, 1990; Hedreen et al., diverse pathological processes. MMPs are zinc-containing 1991). HD is caused by a polyglutamine (polyQ)-encoding CAG proteolytic enzymes secreted to degrade extracellular matrix expansion in the gene for the huntingtin protein (Htt) (The Hunting- proteins (Hornebeck and Lafuma, 1991). An imbalance in ton’s Disease Collaborative Research Group, 1993). Expression MMP/TIMP (tissue inhibitor metalloproteinase) activity is impli- of mutant Htt leads to selective neuronal dysfunction and degen- cated in conditions such as rheumatoid arthritis, kidney disease, eration despite its ubiquitous expression pattern (Bhide et al., cardiovascular disease, and cancer (Baker et al., 2002; 1996; Gourfinkel-An et al., 1997). Truncated mutant Htt, in Konttinen et al., 1999; Ronco et al., 2007). Recent evidence contrast to full-length mutant Htt, induces apoptosis (Martindale has linked inhibition of MMPs to reduced neuronal damage after et al., 1998), and mutant N-terminal Htt fragments have been transient cerebral ischemia (Lee et al., 2009). MMP-3 and observed in human HD tissue and presymptomatic HD mouse MMP-9 knockout mice have significantly decreased striatal models, suggesting that proteolysis is required for disease neuronal cell death after intracerebral hemorrhage (Xue et al., progression (Mende-Mueller et al., 2001; Wang et al., 2008; 2009). Further, intraperitoneal injection of the MMP-3/MMP-9

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 199 Neuron Matrix Metalloproteinases Modify Huntingtin

Figure 1. Western Blot Screen for Protease siRNAs that Reduce the Abundance of the Htt 55 kDa Fragment (A) siRNA/DNA cotransfection of cells was done in 96-well format, and transfected cells were incubated 48 hr before the addition of epoxomicin to stabilize Htt fragments. Cells were lysed and crude lysates transferred to SDS-PAGE loading buffer for subsequent western blot analysis. The first two columns of each plate contained control transfections—antibody specificity control, nontargeting (NT) siRNA/empty vector (gray wells); negative control:, NT siRNA/Htt 138Q full-length (FL) (lavender wells)—and the rest of the plate contained an individual protease siRNA w/Htt138Q FL DNA (green wells). (B) Each of the rows of the 96-well plate transfections in (A) were loaded onto a single SDS-PAGE gel and subjected to western blotting. The antibody 1C2 (MAB1574) detects two robust nonspecific bands (*) in vector only lanes, while the remaining bands in Htt138Q FL lanes are either unique or dramatically enriched. The bands range from the full- length protein (FL) to the 55 kDa fragment of interest (arrowhead). (C) Densitometry spectra of the MMP-10 and NT lanes from the western blot shown in (B). Each gel was subjected to densitometric analysis of bands, and the volume ratio of the 55 kDa to FL was compared to the ratio in the NT control lane to identify candidate proteases. See also Figure S1.

cell death when knocked down in striatal Hdh111Q/111Q cells. We also find that MMP activity is significantly elevated in mouse models of HD and reduced MMP activity inhibitor, SB-3CT, showed a decrease in MMP activity and suppresses Htt-induced neuronal dysfunction in Drosophila. infarct size in the ischemic cortex in a mouse model of stroke These data show that MMPs affect expanded polyQ-Htt proteol- (Gu et al., 2005). While the importance of MMPs in disease has ysis and toxicity, suggesting this family of enzymes may be rele- led to the development of a few highly specific MMP inhibitors, vant therapeutic targets in the disease. further studies are needed to characterize the contribution of MMP family members to normal cell function and disease. RESULTS Given that smaller Htt fragments generally yield greater cellular toxicity (Hackam et al., 1998; Martindale et al., 1998), Identification of Proteases Involved in the Generation we designed a high-throughput western blot-based screen to of the Small N-terminal Mutant Htt Fragment examine the generation of the smallest N-terminal polyQ-con- Given that smaller Htt fragments generally yield greater cellular taining fragment from full-length Htt. We screened a set of toxicity (Hackam et al., 1998; Martindale et al., 1998), we de- 514 small interfering RNA (siRNA) pools targeting the repertoire signed a high-throughput western blot-based screen to examine of all known and predicted protease genes encoded in the the generation of a small N-terminal polyQ-containing fragment human genome to identify those proteases that when inhibited from full-length Htt in a 96-well format (Figure 1A). As shown in reduced Htt proteolysis. Our primary screen identified 41 prote- Figure 1B, this screen allows the analysis of multiple proteolytic ases that alter Htt fragment accumulation, and 11 of these were cleavage products generated from full-length mutant Htt. Mutant confirmed in retesting. Nine of the eleven proteases are ex- Htt and cleavage products were detected with a monoclonal pressed in striatal cells, and their knockdown significantly antibody that recognizes the expanded polyQ stretch in the reduced Htt-mediated striatal cell death in a secondary cellular N-terminus of Htt (1C2). We focused our analysis on the genera- toxicity screen. Furthermore, decreasing the levels of five of tion of the smallest N-terminal polyQ containing Htt fragment these proteases suppressed mutant Htt induced toxicity in a observed, which migrates at 55 kDa (Figure 1B, indicated by Drosophila HD model. arrowhead). siRNAs directed against all 514 known or predicted Of the nine proteases validated by the secondary screen, three human proteases were screened in duplicate for effects on are matrix metalloproteinase family members, suggesting that proteolysis of expanded full-length Htt in HEK293T cells using these enzymes play a role in Htt proteolysis and toxicity. We the western blot assay. In this primary screen, 41 initial hits demonstrate that MMP-10 directly cleaves Htt and reduces were identified that reduced the levels of the smallest N-terminal

200 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

Table 1. Proteases Whose Knockdown Reduced 55 kDa Htt Fragment in Primary Western Blot Screen Gene Symbol GeneID Number Description CAPN5 726 calpain 5 CAPN7 23473 calpain 7 IMP5 162540 intramembrane protease 5 ITGA2B 3674 integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41) KLK10 5655 kallikrein-related peptidase 10 KLK11 11012 kallikrein-related peptidase 11 MMP10 116869 matrix metallopeptidase 10 MMP14 4323 matrix metallopeptidase 14 (membrane-inserted) MMP23B 8510 matrix metallopeptidase 23B RNF128 79589 ring ﬁnger protein 128 SPC18 23478 SEC11 homolog A (S. cerevisiae)

Htt fragment (possible aspartyl Htt cleavage product [Lunkes et al., 2002]) with at least a 30% reduction in expression of the Htt fragment relative to the full-length protein. This analysis was done using densitometry as shown in Figure 1C. The conditions of this assay result in significant knockdown of specific protease targets as demonstrated by western blot analysis of caspases (see Figure S1 available online). Retesting of these 41 primary hits resulted in 11 of the hits Figure 2. Secondary Screen of Striatal Cell-Based Toxicity Assay being confirmed by their consistent decrease in the production (A) PolyQ expansion in mouse knockin striatal cells produces robust caspase of the 55 kDa putative aspartyl Htt cleavage product (Table 1). activation when compared to wild-type controls. Caspase activity was The proteases identified include members of the calpain family measured in striatal Hdh7Q/7Q and Hdh111Q/111Q cells undergoing serum with- (CAPN5 and CAPN7); the signal peptide protease-like IMP5; an drawal for 24 hr. Fold change in activity normalized to nontargeting siRNA (NT) Hdh7Q/7Q Hdh111Q/111Q aminoterminal signal peptide protease (SPC18); members of the in or cells (ANOVA, n = 5, **p < 0.01 or ***p < 0.005). (B) siRNAs targeting 9 of the 11 proteases found as hits in the primary screen secreted serine-protease kallikrein family (KLK10 and KLK11); result in suppression of caspase activation in Hdh111Q/111Q cells. siRNAs to the the transmembrane-E3-ubiquitin ligase, RNF128; the MMP-2 indicated protease or a NT siRNA control were electroporated into interacting integrin, ITGA2B (Choi et al., 2008b); and three Hdh111Q/111Q cells and incubated for 48 hr. Caspase 3/7 activity was then members of the matrix metalloproteinase family (MMP-10, measured 24 hr after serum withdrawal. Fold change in activity normalized 111Q/111Q MMP-14, MMP-23B). to NT siRNA in Hdh , ANOVA analysis, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001. The error bars represent standard deviation. See also Figure S2. Secondary Screen for Modifiers of Htt Toxicity in Immortalized Mouse Striatal Cells There is a body of literature suggesting that truncation of the that they are selectively vulnerable to mitochondrial complex II full-length protein is cytotoxic in all polyQ diseases. For Htt inhibitor-induced cell death through a non-apoptotic pathway protein, the smaller N-terminal truncation products are associ- (Mao et al., 2006; Milakovic and Johnson, 2005). To develop ated with increased toxicity. Since the 55 kDa product is the an assay relevant to cell death, we cultured Hdh7Q/7Q and smallest detectable Htt cleavage product in our western blot Hdh111Q/111Q cells and measured caspase activation 24 hr after analysis, we predict that blocking production of this fragment serum withdrawal (Figure 2A). We found that Hdh111Q/111Q cells will prevent Htt-mediated cell death. To analyze the effect of have a 3.7-fold increase in caspase activation compared to the 11 initial hits identified in our western blot screen, we devel- control Hdh7Q/7Q. Further, siRNA against caspase-3, which oped a Htt-mediated cell death assay relevant to HD. Since HD serves as a positive control in our assay, reduced caspase primarily affects striatal and cortical neurons we used activity by 8.7 fold in Hdh111Q/111Q cells (Figure 2A). immortalized mouse striatal cell Hdh7Q/7Q and Hdh111Q/111Q lines Having established a robust striatal cellular toxicity assay, we for our secondary screen of cellular toxicity, as well as subse- evaluated whether the 11 proteases identified in our primary quent genetic and biochemical studies (Trettel et al., 2000). screen are active in this assay. Intriguingly, siRNA-mediated Previous work in our laboratory has demonstrated that striatal knockdown of 9 of the 11 proteases results in significant cells from mutant Htt knockin mice have altered calcium reduction of toxicity in this cell model (Figure 2B). KLK10 and handling (Oliveira et al., 2006) and other groups have reported KLK11, the two original hits that were negative in this secondary

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 201 Neuron Matrix Metalloproteinases Modify Huntingtin

A assay, are not expressed in striatal cells. We used KLK cDNA transfected Hdh111Q/111Q cells as a positive control to verify KLK antibodies (data not shown). These results suggest that enzymes that promote the produc- *** tion of small N-terminal Htt cleavage products in our western *** blotting assay are likely to modulate HD toxicity. mRNA/ActB expression (ABU) mRNA/ActB expression (ABU) MMP-10 and MMP-14 Are Expressed in Striatal Hdh111Q/111Q Cells B Since three of the nine validated hits (MMP-10, MMP-14, MMP- 23B) are members of the matrix metalloproteinase family and NT siRNA MMP14 siRNA one (ITGA2B) interacts with MMP-2 (Choi et al., 2008b), we chose to further characterize the potential role of this family of enzymes in

Anti-MMP10 Anti-MMP14 HD. Most zinc-containing metalloproteases contain a character- istic HEXXH consensus sequence that coordinates to the zinc. Matrix metalloproteinases are a subfamily of the metzincins, which Anti-tubulin Anti-tubulin share the extended zinc-binding XEXXHXXGXXH consensus motif C and a conserved methionine adjacent to the catalytic zinc. MMPs were discovered by their ability to ‘‘dissolve’’ collagen fibers in * tadpole skin. There are 25 human MMPs (Figure S2) that are struc- turally related zinc- and calcium-dependent enzymes. Some are *** activated intracellularly by furin proteases and many have been implicated in cancer, stroke, and arthritis. To further analyze the effect of MMP knockdown in striatal Caspase 3/7 Activity (dRFU/min/mg) cells, we evaluated whether these proteases were expressed Hdh111Q/111Q in Hdh111Q/111Q cells and if the knockdown occurred in response to the siRNA. Western blotting for MMP-10 and MMP-14 demon- D strated that they are present in Hdh111Q/111Q cells at detectable * levels, and introduction of siRNAs specific to their messenger ** RNAs does cause a significant reduction in MMP mRNA and protein expression (Figures 3A and 3B). We found the level of knockdown correlated with the reduction in caspase activity as titration of siRNA to MMP-10 (or deconvoluted siRNA pools)

Caspase 3/7 Activity (dRFU/min/mg) further reduced protein levels correlating with a reduced caspase-3 activity (data not shown). As shown in Figure 3C, siRNA NNGH directed against MMP-10 or MMP-14 signiﬁcantly reduced E caspase activation in Hdh111Q/111Q cells. We also found that MMP-23B was knocked down by the siRNA treatment and reduced caspase activation in Hdh111Q/111Q cells (data not *** ** shown). The MMP-23B mRNA level of expression in mouse striatal cells and tissue is quite low when compared to MMP-14 (0.00663 ± 0.001 versus 0.5304 ± 0.006; 0.00026 ± 0.00006 versus 0.0078 ± 0.0003). Caspase 3/7 Activity (dRFU/min/mg) Caspase 3/7 Activity (dRFU/min/mg) Next, we tested a pharmacologic inhibitor, NNGH, which is a nonpeptidic, potent inhibitor of MMPs. The crystal structure Figure 3. Knockdown of MMP-10 and MMP-14 and Inhibitors of of the catalytic domain of human matrix metalloproteinase 10 MMP Activity Block Caspase Activation in Striatal Hdh111Q/111Q Cells (A) Quantitative RT-PCR validation of MMP-10 and MMP-14 siRNA knock- has been solved with this molecule (Bertini et al., 2004). In addi- down. t test was performed (p = 0.0004 and p = 0.7E-9, respectively). tion to pharmacologic inhibitors, we tested known endogenous 111Q/111Q (B) Western blot analysis of siRNA targeting on expression of the indicated inhibitors of MMPs-TIMP1 and TIMP3 in Hdh cells. As MMP. shown in Figures 3D and 3E, we found that these inhibitors (C) Effect of siRNA to MMP-10, MMP-14 or nontargeting on activation of cas- blocked Htt-mediated toxicity in Hdh111Q/111Q cells. pase-3 in Hdh111Q/111Q cells during 24 hr serum withdrawal. Caspase activity is in units dRFU/min/mg (ANOVA, n = 5, *p < 0.05 ***p < 0.001). MMP-10 Is Processed in Striatal Hdh111Q/111Q Cells (D) Inhibition of MMPs with NNGH lowers caspase activation in striatal cells (ANOVA analysis, p = 0.027 and 0.005). and MMP Activity Is Increased (E) TIMP1 and TIMP3 overexpression of protein reduces caspase activation in Given the involvement of MMPs in blocking Htt-mediated HD striatal cells (t test was performed, p = 0.3E-6 and 0.007). toxicity, we investigated whether these proteins were processed The error bars represent standard deviation. to their active forms in striatal cells containing mutant Htt. Strik- ingly, we found in striatal Hdh111Q/111Q cells cultured with serum,

202 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

the levels of MMP-10 and MMP-14 were altered when compared to Hdh7Q/7Q (Figure 4A). Further, MMP-10 was processed suggesting activation of the protease in the striatal Hdh111Q/111Q cells as analyzed by western blotting (Figure 4A; 5.7-fold increase in active form in Hdh111Q/111Q versus Hdh7Q/7Q cells, **p < 0.01). The specificity of the MMP antibodies was confirmed by over- expressing MMP-10 and MMP-14 in striatal cells (Figure 4B). MMP-23B levels did not change in striatal Hdh111Q/111Q cells when compared to Hdh7Q/7Q (data not shown). The proteolytic processing of MMP-10 does not necessarily indicate that levels of active MMP have increased in the mutant Htt cells. We therefore evaluated the MMP activity in striatal Hdh7Q/7Q and Hdh111Q/111Q cells. Figure 4C shows that MMP enzymatic activity is significantly increased in striatal Hdh111Q/111Q cells when compared to Hdh7Q/7Q using a fluorogenic substrate assay.

MMPs Are Activated in the R6/2 and YAC128 Mouse Model of HD We next examined whether our cell culture results could be reca- pitulated in mouse models of the disease. We used a full-length Htt (YAC128) and a fragment mouse model (R6/2) of HD. The full- length HD model provides information about the natural history of proteolysis of full-length Htt and the proteases activated. The fragment model can provide information about the amplification and feedback of proteases once a relevant fragment is formed. The MMP enzymatic activity was assayed in the subre- gions of the brain in the YAC128 and R6/2 mouse model of HD. Figure 5A shows that MMP enzymatic activity is indeed increased in 16-month-old YAC128 or 10-week-old R6/2 striatum, with a 1.6- to 1.8-fold increase in MMP activity relative to the control striatum. We also examined the MMP enzymatic activity in another polyQ disease, SCA7 (Mookerjee et al., 2009). As shown in Figure 5A (right panel), MMP enzymatic activity does not increase in SCA7 mice expressing ataxin-7- 92Q. This suggests MMP activation is relevant in HD but may not be a feature of other polyQ diseases. We used zymography to analyze the specific activity of the various MMPs in HD mouse brain, after MMP affinity precipita- tion (using gelatin beads). As shown in Figure 5B, we found elevated MMP activity detected in several bands ranging from 50 kDa to 90 kDa in lysates of the cortex and striatum of R6/2 mouse when compared to controls. The most robust Figure 4. Activation of MMP in Striatal Hdh111Q/111Q Cells Relative increase in R6/2 lysate activity corresponds to the 50 kDa to Hdh7Q/7Q bands (Figure 5B). Since MMP-10 migrates at around 50 kDa (A) Striatal cells were electroporated with the indicated construct and cell in its active form, this result is in agreement with our striatal lysates were analyzed by western blotting with the indicated MMP antibody. cell line studies. We cannot exclude the possibility that other Cells were cultured in the presence of serum. co-migrating MMPs may contribute to the observed activity. (B) Western blotting of striatal cell lysates probed with MMP antibodies. (C) Activity of MMP fluorogenic substrate Mca-Pro-Leu-Dpa-Ala-Arg-NH2 The band at 90 kDa may represent MMP-9 (MW 92 kDa, arrow- (Mca = (7-methoxycoumarin-4-yl)-acetyl; Dpa = N-3(2,3-dinitrophenyl)-L-a- head), a protease activated by MMP-10. b-diaminopropionyl) in striatal Hdh7Q/7Q and Hdh111Q/111Q cell lysates. The activity is expressed in RFU/mg protein (n = 5, t test, **p < 0.01). In Situ Gelatinase Activity The error bars represent standard deviation. Since the MMP activity assay and zymography indicated increased levels of MMP activity in R6/2 and YAC128 lysates, we employed an in situ gelatinase assay to evaluate the levels of Inhibition with the MMP inhibitor 1,10-phenanthroline abolished MMP directly in R6/2 and control tissue sections. In sections the fluorescence signal, confirming that the activity was attribut- from R6/2 mice, we observed increased fluorescence when able to MMPs (Figure S3). Since MMP family members can be compared to control in both the striatum and cortex (Figure 5C). expressed in both glial and neuronal populations, we determined

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 203 Neuron Matrix Metalloproteinases Modify Huntingtin

A Figure 5. MMP Activity Is Elevated in YAC128 and R6/2 Striatal Tissue but Not in a Spinocerebellar * * Ataxin-7 polyQ Mouse Model (A) MMP activity assay with ﬂuorogenic substrate activity measured in units of RFU/mg protein (t test, n = 3, *p < 0.05).

MMP Activity (B) Striatal and cortex tissue immunoprecipitates using gelatin Sepharose 4B beads were resolved on a 10% Control YAC128 Control R6/2 Striatum Cerebellum zymogram gelatin gel and assayed for gelatinase activity. Striatum 15-18 Striatum 10 Weeks 6 Months Arrowhead indicates 90 kDa activity. Months B (C) In situ zymography on 12–13 week control and R6/2 mouse brain tissue. MMP enzyme activity shown by Striatum Cortex a DG gelatin ﬂuorescein conjugate MMP substrate. Control R6/2 Control R6/2 Images are shown at 203 magniﬁcation. 62 – The error bars represent standard deviation. See also

49 – Figure S3.

C Striatum Cortex full-length wild-type or mutant Htt expressed in Control Control cellular lysates is a substrate for MMP-10, but not MMP-14 or MMP-2. A cleavage product appears at 48 kDa originating from normal Htt, and 70 kDa for mutant Htt148Q. In vitro translation of Htt15Q (1-1212) also conﬁrmed that Htt is a substrate for MMP-10 but not MMP-14 or MMP-2 (Figure 6B). All three MMP family members were equally active against the ﬂuo- rescent substrate, Mca-Pro-Leu-Dpa-Ala-Arg- 100 m 100 m NH2, suggesting that Htt is a preferred substrate R6/2 R6/2 for MMP-10 (data not shown). These data indicate that MMP-10 knockdown suppresses Htt toxicity through its direct effect on Htt cleavage while the effect of MMP-14 knockdown is indirect.

MMP-10 Cleaves Htt at Amino Acid 402 MMP-10 appears to cleave Htt in a region closer 100 m 100 m to the N terminus than the caspase and calpain cleavage products (55–72 kDa) (Gafni et al., 2004; Hermel et al., 2004; Wellington et al., 1998) producing a smaller 48 kDa product. the subpopulation that contained MMP activity by costaining with Recent in vitro studies suggest Htt is also cleaved at amino GFAP (glial speciﬁc) or NeuN (neuronal speciﬁc). The majority of acid 167 (Ratovitski et al., 2009). Deletion of the residues at MMP staining is present in neurons, although some MMP activity this site has no effect on the proteolysis of Htt by MMP-10 does colabel GFAP-positive cells (Figure S3). (Figure 6B). Therefore, the site of MMP-10 cleavage is likely between amino acid 167 (Cp2 site) and 469 (calpain site). Direct versus Indirect Effects of MMPs on HD-Mediated Consistent with this, Htt (1-450) is still a substrate for MMP-10 Toxicity (Figure 6C). Knockdown of MMPs suppresses mutant Htt toxicity and To locate the approximate site of cleavage, Htt15Q (1-469) decreases the amount of small Htt fragment accumulation. This and Htt138Q (1-469) were in vitro translated and incubated indicates that MMPs may either cleave mutant Htt directly or with MMP-10. As shown in Figure 6D, MMP-10 addition to modulate events upstream or downstream of Htt cleavage. For Htt15Q (1-469) and Htt138Q (1-469) generates a 48 kDa and example, in HIV, MMP-2 cleaves stromal cell-derived factor 70 kDa fragment, respectively. Htt15Q (1-167), Htt15Q (1-329), (SDF)-1a to activate neuronal cell death (Vergote et al., 2006). Htt15Q (1-378), Htt15Q (1-414), and Htt138Q (1-167) serve as To determine whether Htt is a direct substrate for MMP-10 or size controls. Analysis of the molecular mass of the Htt15Q MMP-14, we incubated cell lysates expressing Htt with the cleavage product suggests cleavage occurs around amino recombinant MMPs. As a control, we included MMP-2, an acid 414 of Htt. Examination of the amino acid sequence of Htt MMP family member not found to alter HD cellular toxicity or in this region, and comparison to known MMP substrates, proteolysis in our original screen. As shown in Figure 6A, suggests a possible cleavage site at amino acid 402. To test

204 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

ABC

DEF

Figure 6. MMP10 Directly Cleaves Htt (A) Full-length Htt23Q and Htt148Q cellular 293T lysates (with protease inhibitors) were treated with recombinant MMP-2, MMP-10, or MMP-14 and western blot was probed with Htt N-terminal antibody. (B) In vitro translated Htt15Q (1-1212) and Htt15Q (1-1212) D167–170 mutants were treated with recombinant MMP-2, MMP-10, or MMP-14. A Htt cleavage product of 45 kDa appears when MMP-10 is added and is not affected by deletion of amino acids 167–170. (C) Htt23Q (1-450) and Htt57Q (1-450) cellular lysates were treated with recombinant MMP-10 and western blot was probed with anti-6HisGS (tag at N terminus of Htt). (D) In vitro translation of Htt15Q (1-469), Htt15Q (1-414), and Htt15Q (1-378) demonstrates that cleavage occurs between amino acids 378 and 414. (E) In vitro translation of a Htt15Q (1-469) deletion mutant (D402-403 or DGI) reduces the MMP-10 product. (F) Control and HD postmortem tissue lysates from caudate were analyzed by western blotting. Blots were probed with anti-huntingtin 115–129 antibody. The size of the fragment is similar to that generated by MMP-10 treatment of HD lysates (with protease inhibitors) shown in lane 3 and 4. TBP was utilized as a loading control. (G) Immunofluorescence of striatal Hdh111Q/111Q cells labeled with anti-huntingtin 115–129 antibody (green) and anti-MMP-10 antibody (red). Left panel cultured in serum; right cultured in serum-free media. Cells are shown at 1263 magnification and arrows indicate cell analyzed and colocalization. Arrowheads point to MMP-10 colocalization. Quantitative analysis of immunofluorescence via Imaris software indicates significant colocalization in cells as shown in right panel (t test, **p < 0.01). The error bars represent standard deviation. See also Figure S4. this possibility, we carried out deletion analysis on this site. As size controls. Htt15Q (1-402) matches the molecular mass of shown in Figure 6E, deletion of amino acids 402–403 (DGI) of the MMP-10-generated Htt cleavage product. Htt results in a protein resistant to proteolysis by MMP-10. To confirm that the cleavage product is generated in vivo, HD Htt15Q (1-414), Htt15Q (1-402) and Htt15Q (1-378) serve as post-mortem cellular lysate were compared to control lysate and

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 205 Neuron Matrix Metalloproteinases Modify Huntingtin

analyzed by western blotting. As shown in Figure 6F, a Htt the retina when compared to controls (Figure S5). Animals cleavage product around 45–55 kDa is evident in the HD tissue expressing NT-Htt[128Q] and with decreased levels of CalpA, and is consistent with the size of the products in our in vitro Sol, and Dm2-MMP showed improved retinal integrity and assays (accounting for a CAG repeat in the patient of 42). improved thickness when compared to animals expressing Consistent with this, when we treated HD postmortem cellular NT-Htt[128Q] alone. lysates with MMP-10 a cleavage product increased at 50 kDa (Figure 6F). Further, recent analysis of the HD150 knockin mice DISCUSSION suggests Htt fragments of this size are present in striatal and cortical lysates (Landles et al., 2010). We have carried out an unbiased RNAi screen to identify Since Htt is likely to be a substrate via intracellular cleavage by proteases that when knocked down reduced the production of MMP-10, we evaluated localization of endogenous Htt and toxic N-terminal Htt proteolytic fragments. We screened 514 siR- MMP-10. As shown in Figure 6G, Htt and MMP-10 colocalize NAs targeting all known and predicted human proteases and in discrete punctate structures (left panel). Further, in cells found 11 proteases that altered the level of the smallest undergoing cell death Htt and MMP-10 show enhanced colocal- N-terminal mutant Htt protein detected by the 1C2 anti-polyQ ization (right panel). Quantification of colocalization is shown in antibody. The proteases identified include members of the the bar graph of Figure 6G. The colocalization does not result calpain family (CAPN5 and CAPN7); the signal peptide from a direct protein-protein interaction as we did not find protease-like IMP5 and an amino-terminal signal peptide MMP-10 and Htt coimmunoprecipitate (Figure S4). protease (SPC18); members of the secreted serine-protease kallikrein family (KLK10 and KLK11); the transmembrane-E3- Reduced Activities of MMP and Other Proteases ubiquitin ligase, RNF128; the MMP-2 interacting integrin, Identified in the siRNA Screens Improve Neuronal ITGA2B (Choi et al., 2008b); and three members of the matrix Function in a Drosophila Model of HD metalloproteinase family (MMP-10, MMP-14, MMP-23B). The data presented above suggest that reducing the activity Our results are consistent with the prior observations demon- of MMPs, and perhaps other proteases, may ameliorate Htt- strating that calpain family members are involved in the induced neuronal dysfunction in vivo. To test this idea, we proteolysis of Htt and pathogenesis of HD (Gafni and Ellerby, used a behavioral readout in a HD Drosophila model expressing 2002; Gafni et al., 2004; Kim et al., 2001). Both CAPN5 and the first 336 amino acids of the human protein including 128 CAPN7 levels are increased in HD mouse models and post-mor- glutamines in exon 1. In this model system, neuron-specific tem tissue. Several other interesting proteases were identified in expression of expanded Htt leads to quantifiable progressive our studies. For example, IMP5, a signal peptide peptidase (SPP) motor deficits (Kaltenbach et al., 2007). In contrast to the is an unusual aspartyl protease that mediates clearance of signal mammalian genome that contains 25 MMPs with partially over- peptides by proteolysis within the endoplasmic reticulum (ER) lapping functions, Drosophila only has two MMP family (Krawitz et al., 2005). Like presenilins, the proteolytically active members, Dm1-MMP and Dm2-MMP. Although there are no subunit of the gamma-secretase complex, SPP contains clear orthologous relationships between the Drosophila and a critical GXGD motif in its C-terminal catalytic center. Htt is mammalian genes, they are clearly evolutionarily conserved as associated with the ER, and perhaps IMP5 plays a role in Htt evidenced by their domain structure, amino acid sequence processing in this organelle (Atwal and Truant, 2008). Currently similarities, and functional interactions (Llano et al., 2000; the substrates and mechanism for these proteases are unknown. Page-McCaw, 2003, 2008; Wei et al., 2003). Of the two Another identified protease, RNF128, is a ubiquitin E3 ligase, that Drosophila genes Dm2-MMP is expressed in the postembryonic promotes proteasomal degradation via Lys-48 linkages by brain (Page-McCaw et al., 2003), while Dm1-MMP expression capture of transmembrane substrates (Lineberry et al., 2008). was not detected in the adult (Llano et al., 2000). Thus, we asked Further studies will be required to determine if Htt is a substrate whether genetically reducing the activity of Dm2-MMP leads to for RNF128 and how this enzyme alters proteolysis of Htt as improvement of Htt-induced motor dysfunction. We found that observed in our screen. Decreasing the levels of these four heterozygous loss-of-function alleles of Dm2-MMP show robust proteases (CAPN5, CAPN7, IMP5, and RNF18) suppresses and significant effects in improving the motor performance of HD Htt-induced toxicity in mouse striatal cells. Furthermore, knock- flies. Importantly, this observation was reproduced with two down of their Drosophila homologs suppresses neuronal different Dm2-MMP alleles indicating that the effect is caused dysfunction in a fly HD model. This underlines their potential as by reduced function of Dm2-MMP, and not by genetic back- therapeutic targets for HD. ground effects (Figures 7A, 7I, and 7J). Figure 7 shows as well Over 36% of the proteases identified in our screen were matrix that partial loss of function in Drosophila homologs of CAPN5, metalloproteinase family members, and therefore we focused CAPN7, IMP5, and RNF128 also ameliorate motor deficits in our analysis on these proteases in HD. In humans, there are the HD Drosophila model. These data support the idea that 187 metalloproteases, and this represents the largest of the MMPs and perhaps other proteases characterized here may five protease classes (Hornebeck and Lafuma, 1991; Malemud, be therapeutic targets in HD. 2006). There are 25 human MMPs and four TIMPs. MMPs are In addition to the behavioral readout, we also tested for photo- traditionally thought to degrade the extracellular matrix, but receptor degeneration in these flies (Figure S5). Expression of emerging technology suggests there are many other substrates NT-Htt[128Q] in the eye causes severe photoreceptor degener- for MMPs such as chemokines, cell receptors, cyotokines, ation, tissue loss and significant shortening in the thickness of growth factors and fas ligands. We found that the knockdown

206 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

Figure 7. Reduced Activity of Proteases Improves Motor Dysfunction in HD Drosophila Model (A) Table summarizing the effects caused by modulating the levels of the indicated proteases on NT-Htt[128Q]-induced motor performance in Drosophila. Columns 1 and 2 list the human proteases and Drosophila homologs; column 3 shows the amino acid sequence similarity as the blast E value; column 4 indicates the specific allele tested; column 5 shows the allele class: inducible RNAi constructs (RNAi), loss of function caused by insertion of transposable element (LOF) or overexpression caused by an activating transposable element (OE); column 6 reveals the suppressor (S) or enhancer (E) effect of each allele on NT-Htt[128Q]- induced motor deficits. (B–K) Quantification of motor performance as a function of age in flies of the indicated genotypes using a climbing assay. Control animals expressing just the Elav-Gal4 driver perform well in the climbing assay beyond 14 days (control-blue dashed line). Flies expressing NT-Htt[128Q] in the nervous system either alone or together with a control RNAi, show progressive motor dysfunction when compared with controls (NT-Htt[128Q] and NT-Htt[128Q]/UAS-whiteRNAi, respectively. Black dashed lines). Flies expressing NT-Htt[128Q] in the nervous system but with decreased levels of the proteases: CalpA (CAPN5 homolog, B–C); Sol (CAPN7 homolog, E); Spp (IMP5 homolog, F); CG17370 (IMP5 homolog, G–H); Dm2-MMP (MMP homolog, I–J) or Gol (RNF18 homolog, K) show improved motor performance (red solid line in Band C and E–H). In contrast, flies expressing NT-Htt[128Q] with increased levels of the protease Sol (CAPN7 homolog) display a worse motor performance (red solid line in D). Data was analyzed using ANOVA followed by Tukey’s hsd. Error bars represent SEM. *p < 0.01, **p < 0.001, ***p < 0.0001. Flies were raised at 26.5C. See also Figure S5. of the MMP-2 interacting integrin ITGA2B (Choi et al., 2008b) and known to be upregulated in stroke and is present in neurons three members of the matrix metalloproteinase family (MMP-10, (Cuadrado et al., 2009). MMP-14, MMP-23) suppressed toxicity in an immortalized stria- The mechanism by which MMPs suppress HD-mediated tal cell line harboring a knock-in of mutant Htt. We show that toxicity could be through the known function of MMPs in MMP activity is increased in cell culture and animal models of targeting the extracellular matrix or cytokines (see Figure 8 for HD. Furthermore, MMP-10 is proteolytically processed and potential mechanisms). However, our screen was designed to activated in these models. Relevant to our studies, MMP-10 is identify proteases that when knocked down would reduce the

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 207 Neuron Matrix Metalloproteinases Modify Huntingtin

Figure 8. Model of Possible Mechanisms for Htt or Neurotoxicity of MMPs in Huntington’s Neuron Disease MMP In HD striatal cells, MMP-10 and MMP-14 are activated. Upon activation, MMPs may cause neuro- Neurotoxicity toxicity at different cellular locations/substrates in our model. Htt may be processed directly or Other Htt substrates indirectly by MMPs (through activation of other proteases). In striatal neurons, MMP-10 may process mutant Htt into a toxic fragment intracellularly MMP and possibly processing other cellular substates. Proteolysis of these substrates would activate Glial cell or cell death. On the outside of the cell (MMP is mem- Htt brane bound or secreted), proteolysis of extracellular substrates could result in processing of MMP substrates involved in inﬂammation. This could also occur in glia as depicted in our model.

Extracellular MMP MMP substrates Striatal MMPs to be activated in cellular and neuron animal HD models. Knockdown of par- Neurotoxicity ticular MMP family members reduced MMP mutant Htt-mediated cell death in an immortalized striatal cell line and signifi- MMP cantly improved motor deficits caused by neuronal expression of polyQ-expanded Htt in Drosophila. These data suggest Glial cell that selective inhibition of the implicated MMP family members should be consid- ered for developing novel therapeutics proteolysis of mutant Htt. In order to determine the mechanism for HD. For example, MMP-9 knockout mice show improved for MMPs in HD, we evaluated whether Htt is a substrate for outcomes after cerebral ischemia (Asahi et al., 2000). This may particular MMP family members. We found that MMP-10 cleaves be directly relevant to HD as a recent analysis of postmortem Htt directly whereas MMP-14 and MMP-2 do not. Inhibition of HD revealed MMP-9 is upregulated in HD brain and absent in MMP-10 by siRNA-mediated knockdown reduces the genera- age-matched controls (Silvestroni et al., 2009). AG3340 (prino- tion of the smallest detectable N-terminal polyQ-containing mastat), a small molecule hydroxamate-based inhibitor of fragment of Htt in cell culture. We mapped the cleavage site of MMPs, was examined in a model of chronic cerebral hypoperfu- Htt generated by MMP-10 to amino acid 402. It is noteworthy sion and is neuroprotective in adult rats and mice when adminis- that the MMP-10 cleavage site in Htt is highly conserved, with tered just before insult (Cai et al., 2006). Reduced activation of (S/T)XXGG(I/L) being present from human to Fugu. These results astrocytes and microglia was also associated with retained suggest that knockdown of MMP-10 reduces HD toxicity blood-brain barrier integrity in these studies. In addition, in through directly effecting the proteolysis of Htt. For the other a mouse model of middle cerebral artery occlusion, the gelati- MMP family members, known mechanisms of cleavage of nase-selective compound SB-3CT reduced infarct volume extracellular matrix or cytokines is likely to play a significant when administered starting at either 2 or 6 hr after insult (Gu role in reducing HD-mediated toxicity. et al., 2005). Recent evidence suggests that some matrix metalloprotei- Taken together, our results suggest that general inhibition of nase family members such as MMP-3 function inside the cell MMPs may be of therapeutic benefit in Huntington’s disease during neuronal cell death (Choi et al., 2008a). While MMPs are and that specific inhibitors of MMP-10 may be particularly generally thought to be secreted as proenzymes and processed relevant to disease treatment. to their active forms outside the cell, we found that MMP-10 was activated inside the cell. This has been reported for other MMPs EXPERIMENTAL PROCEDURES (Luo et al., 2002; Pei and Weiss, 1995, 1996). Furthermore, we found that MMP-10 and Htt colocalize, suggesting the possibility Plasmid Constructs that cleavage occurs intracellularly. Htt is known to associate The full-length expanded polyQ Htt expression construct, Htt138Q, and with membranes and has a site of palmitoylation that would allow pcDNA 3.1 (Invitrogen), were used for the western blotting screen (Martindale et al., 1998). For evaluating MMP proteolysis of Htt with recombinant enzymes, proteolysis to occur at the membrane surface (Kegel et al., human Htt constructs expressing the first 469, 414, 402, 378, 329, and 167 2009a, 2009b; Yanai et al., 2006). amino acids were made by site-directed mutagenesis of the Htt 15Q and Activation of MMPs is known to occur in diseases including 138Q (1-1212) constructs (Wellington et al., 2000). Amino acids 167 to 170 stroke, cancer and HIV (Malemud, 2006). In our study, we found were deleted in Htt 15Q (1-1212) and 402 to 403 were deleted in Htt 15Q

208 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

(1-469) to help map the MMP cleavage site. Primers for the 469 stop construct is a fluorescence-quenched Rhodamine-DEVD conjugate ((zDEVD)2-Rhoda- were F, 50-CAGCAGCTCTGCCTTAACATAGTCAGTGAAGGATGAGATC-30, mine 110) that fluoresces (Ex485nm/Em530nm) upon cleavage of DEVD by and R, 50-GATCTCATCCTTCAC TGACTATGTTAAGGCAGAGCTGCTG-30; caspase 3/7. Fluorescence was read for 1 hr at 37C using a Fusion-Alpha 414 stop construct were F, 50-GCTAAGGAGGA GTCTGGTTGACGAAGCCGT FP HT (PerkinElmer). Toxicity is represented as caspase 3/7 activity in AGTGGGAG-30, and R, 50-CTCCCACTACGGCTTCGTC AACCAGACTCC dRFU/min/mg protein. TCCTTAGC-30; 402 stop construct were F, 50-CTGACCGCAGTCGGGG 0 0 GCTAGGGGCAGCTCACCGCTGCTAAG-3 , and R, 5 -CTTAGCAGCGGTGA Hdh7Q/7Q Hdh111Q/111Q 0 Western Blotting of Striatal and using siRNA GCTGCCCCTAGCC CCCGACTGCGGTCAG-3 ; 378 stop construct were Directed against Target Proteases F, 50-CAATGTTGTGACCGGAGCCTAGGAG CTGTTGCAGCAGCTC-30, and Dharmacon siGENOME SMARTpools (Thermo Scientific) were used for siRNA R, 50-GAGCTGCTGCAACAGCTCC-TAGGCTCCGGTCACAACATTG-30; 329 disruption of CASP3 (M-043042), CAPN5 (M-042053), CAPN7 (M-043031), stop construct were F, 50-GGTGCCCTTGCTGCAGCAGTAGGTCAAGGACAC IMP5 (M-054596), ITGA2B (M-046584), KLK10 (M-062688), KLK11 AAGCCTG-30, and R, 50-CAGGCTTGTGTCCTTGACCTACTGCTGCAGCAAG (M-043814), MMP-10 (M-049762), MMP-14 (M-062241), MMP-23 (M- GGCACC-30; 167 stop construct were F, 50-GATTCTAATCTTCCAAGGTAGC 0 0 047760), RNF128 (M-060871), SPC18 (M-056742), and nontargeting (D- AGCTCGAGCTCTATAAGG-3 , and R: 5 -CCTTAT AGAGCTCGAGCT-GCT 7Q/7Q 111Q/111Q 001206). Striatal Hdh and Hdh cells were maintained at 33 C ACCTTGGAAGATTAGAATC-30; D167–170 were F, 50-CAAAGCTTTGATGG in a humidified atmosphere of 95% air and 5% CO2, in DMEM containing A-TTCTAATCTTCCAGAGCTCTATAAGG-30, and R, 50-CCTTATAGAGCT 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. RNAi treatment 0 D CTGG-AAGATTAGAATCCATCAAAGCTTTG-3 ; and 402–403 were F, 3 6 0 0 0 of cells was as follows: 1 10 cells per siRNA treatment were collected 5 -ccctgaccgcagt cggcggccagctcaccgctgctaaggag-3 , and R, 5 -CTCCTTAG 5 and nucleofected using Kit L (Amaxa) with 3 mg of siRNA, and then 6 3 10 CAGCGGTGAGCTG GCCGCCGACTGCGGTCAGGG-30. Polymerase chain cells/well were plated on 6-well plates. After 48 hr incubation, cells were serum reactions used 100 ng of DNA, 5.0 mlof103 Pfu buffer (Stratagene), 0.2 mM starved for 24 hr followed by harvesting for analysis by western blotting. dNTPs (Promega), 125 ng each of forward and reverse primers (Integrated For western blotting without siRNAs the cells were treated with and without DNA Technologies), 5% Me SO, and 1 ml Pfu polymerase (Stratagene) for 2 serum. 18 cycles at 96C for 1 min, 55C for 1 min, and 68C for 24 min, then 68C Cells were resuspended in M-PER with protease inhibitors (1 tablet/10 ml, for 7 min. Plasmids were DpnI (Promega)-treated, transformed into XL1-Blue Complete Mini, EDTA-free, Roche). Whole-cell lysates were sonicated 5 3 supercompetent cells (Stratagene), and purified using the QIAprep Spin Mini- 5 s pulses at 40 mA. Samples were centrifuged at 22K rpm and supernatant prep Kit (QIAGEN). Mutations and CAG length were confirmed by DNA was collected for western blotting. 43 LDS sample buffer (Invitrogen) and sequencing. Constructs containing 6his-Htt23Q (1-450) and 6his-Htt55Q 1 ml of 1M DTT were added to 20–40 mg total protein and boiled for 10 min. (1-450) were used for western analysis (Kaltenbach et al., 2007). Samples were resolved by SDS-PAGE using 4%–12% NuPage Bis-Tris gels under reducing conditions in MES running buffer. Gels were run at a constant Htt Proteolysis Western Blot Primary Screen 200V for 1 hr, transferred to 0.45 mm nitrocellulose membranes in 13 NuPage We screened Dharmacon’s siGENOME SMARTPOOL Protease Set consisting transfer buffer for 8 hr at 20V. Membranes were incubated in TBS with 0.1% of 514 protease siRNA pools of four duplexes for their ability to reduce the Tween 20 (TBS-T), 5% non-fat milk for 1 hr. Primary antibodies anti-MMP- abundance of a mutant Htt N-terminal fragment. Human embryonic kidney 10 (1:5000, Abcam ab28205), MMP-14 (1:2000, Abcam ab51074), MMP-23 293T (HEK293T) cells were cultured in Dulbecco’s modified eagle medium (1: 500, Abcam ab5314); anti-polyQ (1:2000, Chemicon, MAB1574); and (DMEM; Mediatech) containing penicillin/streptomycin (100 units/ml/ anti-Htt (1:500, MAB2166; 1:1000, MAB5490; 1:1000, MAB5492; 1:100 m 100 g/ml) and 10% heat-inactivated fetal bovine serum (FBS) (GIBCO). Cells MAB5374, all from Chemicon); anti-tubulin (1:1000, Sigma); and anti-b-actin 3 were seeded in DMEM with 10% serum without penicillin/streptomycin at 5 (1:1000, Cell Signaling) were diluted into TBS with 0.1% Tween 20 (TBS-T), 4 10 cells/well in collagen-coated 96-well plates (BD, Biocoat) and incubated at 5% non-fat milk for 1 hr. Blots were developed using Pierce ECL (Thermo 37 C for 24 hr. Lipofectamine 2000 (Invitrogen) was used for transient transfec- Scientific). Band intensity was quantified using ImageQuant TL v2005. tions according to manufacturer’s instructions. Cells were transfected with 80 ng of DsRed DNA, 0.32 mg pcI-Htt138Q DNA, 136 nM siRNA and 1 mg Lip- ofectamine 2000 in 25 ml DMEM lacking serum or penicillin/streptomycin. After RT-PCR of Striatal Cell Knockdown and Mouse Tissue mRNA a 48 hr incubation at 37C, media was replaced with DMEM containing 10% Total RNA was isolated from electroporated striatal cells (1 well of a 6-well FBS and 1 mM epoxomicin and incubated for an additional 24 hr. Cells were plate) or striatal mouse tissue with the RNeasy Mini Kit (QIAGEN) according lysed directly in wells with 25 ml M-PER (Thermo Scientific) containing protease to the manufacturer’s instructions. One microgram of RNA was converted to inhibitors (1 tablet/10 ml, Complete Mini, EDTA-free, Roche) and RQ1 DNase cDNA by using the Message Sensor RT kit (Applied Biosystems). Real-time quantitative PCR (qPCR) was performed with either Universal Probe Library (Promega) and 1 mM MgCl2. Plates were shaken at 700 rpm for 10 min. To 10 ml of lysate, 6 mlof43 LDS loading buffer (Invitrogen) and 1 ml of 1M DTT was dye (UPL from Roche) or SYBR Green (Applied Biosystems) on the LightCycler added and heated to 99C for 5 min. Lysate proteins were resolved on 4%– 480 system (Roche). For quantification the threshold cycle Cp of each ampli- 12% bis-tris gels, transferred to nitrocellulose membrane, and probed with fication was determined by the second derivative analysis provided by the -DDCp monoclonal antibody MAB1574 (1C2, 1:2000, Chemicon). Multiple film expo- LightCycler 480 software and the 2 method was used to determine the sures were collected for each blot, and nonsaturated films were scanned and relative expression level of each gene normalized against the house-keeping b analyzed by densitometry using ImageQuant TL. Hits were identified as those gene -actin. The specificity of each pair of primers was tested by comparing siRNAs which reduced the amount of the 55 kDa band (relative to the FL Htt) to a negative control sample of water on both quantification analysis and high by 30% or more in duplicate. The 41 siRNAs that met these criteria were then resolution melting curve analysis. The primers used are as follows: MMP10 F, 0 0 0 retested in triplicate. 5 -ATGTTCTGTGGCTCCGGATGAG-3 ,R,5-TGTGCTCAGGTGATGCTTT GTG-30; MMP14 F, 50-AACTTCGTGTTGCCTGATGA-30,R,50-TTTGTGGG TGACCCTGACTT-30; MMP23 F, 50-GCACTGTCCCAGGATGAACT-30,R,50-C Secondary Screen for Caspase 3/7 Activity Suppression in Striatal 0 Hdh7Q/7Q and Hdh111Q/111Q Cells CCAGGATGCACACACAA-3 . Striatal cell lines cells were nucleofected with siRNA as described below, plated at 5 3 104 cells/well on collagen-coated 96-well plates, and incubated MMP Cell Activity Assays for 48 hr in DMEM containing 10% FBS and 100U/ml/100 mg/ml penicillin/ Striatal Hdh7Q/7Q and Hdh111Q/111Q cell pellets or mouse striatal tissue were streptomycin, followed by serum-deprivation for 24 hr before assaying. Cells lysed in M-PER or T-PER (Thermo Scientific), respectively. MMP enzyme were lysed in 50 ml of a 50/50 mixture of DMEM/Apo Lysis Buffer (Cell Tech- activity was measured using the MMP assay kit (Biomol) per the manufacturer’s nology Inc). Aliquots (10 ml) were taken for protein quantification by the BCA protocol with the fluorogenic substrate Mca-Pro-Leu-Dpa-Ala-Arg-NH2 (Mca = method (Pierce) before the addition of DMEM/Apo Lysis Buffer with 20 mM (7-methoxycoumarin-4-yl)-acetyl; Dpa = N-3(2,3-dinitrophenyl)-L-a-b-diami- DTT and 2% APO 3/7 HTS Substrate (Cell Technology, Inc.). The substrate nopropionyl). Fluorescence was normalized to total protein in the lysate.

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 209 Neuron Matrix Metalloproteinases Modify Huntingtin

Gelatinase Gel Zymography SUPPLEMENTAL INFORMATION Gelatinase activity from cortex and striatum of R6/2 and control mouse tissue lysates (T-PER) was measured following affinity purification. Briefly, 500 mgof Supplemental Information includes five figures and Supplemental Experi- total protein was added to 500 ml lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM mental Procedures and can be found with this article online at doi:10.1016/j.

NaCl, 5 mM CaCl2, 0.05% Brij-35, 1% Triton X-100, and 0.02% NaN3) and neuron.2010.06.021. affinity purified over gelatin Sepharose (GE Healthcare). MMP protein was eluted with 10% DMSO in PBS. One hundred fifty micrograms of purified ACKNOWLEDGMENTS lysate was added to three microliters of 63 SDS buffer and resolved on a 10% zymogram gel (Invitrogen) containing 0.1% gelatin. Following electro- This work was supported by NIH (NS040251 to L.M.E.), (NS042179 to J.B.), phoresis, gels were incubated in 2.7% Triton X-100 at room temperature for CHDI (to R.E.H., L.M.E., and J.B.), T32 training grant AG000266 (J.P.M.), and 30 min then placed in developing buffer, 50 mM Tris-HCl (pH 7.6), 200 mM Nathan Shock P30AG025708. We thank Danielle Crippen for training in immu- NaCl, 5 mM CaCl2, and 0.05% Brij-35, for 72 hr at 37 C. After developing, nohistochemistry and Espen Walker, University of New Mexico, for assistance gels were stained in SimplyBlue (Invitrogen) for 1 hr at room temperature in providing in situ zymography protocol. Hdh7Q/7Q and Hdh111Q/111Q cells and destained in ddH2O overnight. Gelatinase activity was detected as a were generously provided by Dr. Marcy MacDonald (Massachusetts General clearing of the blue-stained gelatin. Mouse tissue was collected in accordance Hospital). I.A.-R. was funded by a postdoctoral fellowship from the HDF. We protocols approved by IACUC. thank Doug Beckner for technical help on graphics.

Accepted: June 15, 2010 In Situ Zymography, Immunohistochemistry of R6/2, and Control Published: July 28, 2010 Brain Sections R6/2 and control mice were perfused with ice cold PBS (pH 7.4), and brain REFERENCES tissue was collected and immediately frozen on dry ice. Frozen 20 mm sagittal sections were mounted on slides and quickly exposed to zymography solution Albin, R.L. (1995). Selective neurodegeneration in Huntington’s disease. Ann. of pH 7.4 containing 50 mM Tris-HCL, 150 mM NaCl, 5 mM CaCl , 0.3 mM 2 Neurol. 38, 835–836. NaN3, and 40 mg DQ gelatin (Molecular Probes, Eugene, OR) overnight at Asahi, M., Asahi, K., Jung, J.C., del Zoppo, G.J., Fini, M.E., and Lo, E.H. (2000). 37 C. Control slides included 1 mM 1-10-phenanthroline MMP inhibitor. Reac- Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of tion products were visualized on a confocal microscope (LSM510, Zeiss). gene knockout and enzyme inhibition with BB-94. J. Cereb. Blood Flow Metab. In situ zymography sections were fixed in 4% PFA in PBS, permeabilized in 20, 1681–1689. 1% Tween in 13 TBS, blocked in 10% normal donkey serum with chicken anti- mouse IgG at 1:500. Primary antibodies were diluted in 1% BSA in 13 TBS and Atwal, R.S., and Truant, R. (2008). A stress sensitive ER membrane-associa- incubated 24 hr at 4C. All washes were done 3 3 10 min in 1X TBS. Secondary tion domain in Huntingtin protein defines a potential role for Huntingtin in the antibody was diluted in 1% BSA in 13 TBS and applied to slices for 1 hr. regulation of autophagy. Autophagy 4, 91–93. MAB5374 Htt(82-150) antibody (1:50, Chemicon) was labeled with Alexa 647 Baker, A.H., Edwards, D.R., and Murphy, G. (2002). Metalloproteinase inhibi- donkey anti-mouse (Molecular Probes); and GFAP (1:500, Sigma) was labeled tors: biological actions and therapeutic opportunities. J. Cell Sci. 115, with Alexa 555 donkey anti-rabbit (Molecular Probes). Following incubation in 3719–3727. secondary antibody, slices were washed and mounted in Prolong Gold (Invi- Bertini, I., Calderone, V., Fragai, M., Luchinat, C., Mangani, S., and Terni, B. trogen) with DAPI. (2004). Crystal structure of the catalytic domain of human matrix metalloproteinase 10. J. Mol. Biol. 336, 707–716. In Vitro Protein Synthesis and Cleavage Bhide, P.G., Day, M., Sapp, E., Schwarz, C., Sheth, A., Kim, J., Young, A.B., Htt15Q (1-1212), Htt15Q (1-469), Htt138Q (1-469), Htt 15Q (1-414), Htt 15Q (1- Penney, J., Golden, J., Aronin, N., and DiFiglia, M. (1996). Expression of normal 16 402), Htt 15Q (1-378), Htt 15Q (1-329), Htt15Q (1-167), Htt138Q (1-167), and mutant huntingtin in the developing brain. J. Neurosci. , 5523–5535. Htt15Q (1-1212) D167–170 and Htt 15Q(1-469) D402–403 constructs were Cai, Z., Lin, S., Fan, L.W., Pang, Y., and Rhodes, P.G. (2006). Minocycline alle- translated with TNTCoupled Reticulocyte Lysate Systems kit (Promega). Three viates hypoxic-ischemic injury to developing oligodendrocytes in the neonatal microliters of translation product was diluted in M-PER and 10 U of the cata- rat brain. Neuroscience 137, 425–435. lytic domain of MMP-2, MMP-10, or MMP-14 (Biomol) were added and incu- Choi, D.H., Kim, E.M., Son, H.J., Joh, T.H., Kim, Y.S., Kim, D., Flint Beal, M., bated for 2 hr at 37 C. Following addition of SDS and DTT, samples were and Hwang, O. (2008a). A novel intracellular role of matrix metalloprotei- heated to 90 C for 10 min and run on 12% Bis-Tris gels. Gels were fixed nase-3 during apoptosis of dopaminergic cells. J. Neurochem. 106, 405–415. and dried before exposing to film. Choi, W.S., Jeon, O.H., Kim, H.H., and Kim, D.S. (2008b). MMP-2 regulates human platelet activation by interacting with integrin alphaIIbbeta3. J. Thromb. 6 Immunocytochemistry of Striatal Hdh111Q/111Q Cells Haemost. , 517–523. Immunocytochemistry was performed on Hdh111Q/111Q cells cultured with Cuadrado, E., Rosell, A., Penalba, A., Slevin, M., Alvarez-Sabıń, J., Ortega- serum or 24 hr serum depravation. Cells were fixed in 4% PFA in PBS for Aznar, A., and Montaner, J. (2009). Vascular MMP-9/TIMP-2 and neuronal 20 min and washed 5 min in 13 PBS and 23 5 min in 13 TBS. Blocking, MMP-10 up-regulation in human brain after stroke: a combined laser microdis- primary antibodies, secondary antibodies, and washes were applied accord- section and protein array study. J. Proteome Res. 8, 3191–3197. ing to in situ procedure described above. MAB5490 antibody Htt (115-129) Cudkowicz, M., and Kowall, N.W. (1990). Degeneration of pyramidal projection (1:500, Chemicon) was labeled with Alexa 555 donkey anti-mouse (Molecular neurons in Huntington’s disease cortex. Ann. Neurol. 27, 200–204. Probes) and MMP10 antibody (1:2000, Abcam) was labeled with Alexa 488 Gafni, J., and Ellerby, L.M. (2002). Calpain activation in Huntington’s disease. 3 3 donkey anti-rabbit. Cells were washed 3 5 min in 1 TBS and mounted in J. Neurosci. 22, 4842–4849. Prolong Gold (Invitrogen) with DAPI. Gafni, J., Hermel, E., Young, J.E., Wellington, C.L., Hayden, M.R., and Ellerby, L.M. (2004). Inhibition of calpain cleavage of huntingtin reduces toxicity: accu- Drosophila Motor Performance Assay mulation of calpain/caspase fragments in the nucleus. J. Biol. Chem. 279, Strains were obtained from either the Bloomington Stock Center (http:// 20211–20220. flystocks.bio.indiana.edu) or the Vienna Drosophila RNAi Center (http:// Goldberg, Y.P., Nicholson, D.W., Rasper, D.M., Kalchman, M.A., Koide, H.B., stockcenter.vdrc.at/control/main). Motor performance test are described in Graham, R.K., Bromm, M., Kazemi-Esfarjani, P., Thornberry, N.A., Vaillan- the Supplemental Experimental Procedures. court, J.P., and Hayden, M.R. (1996). Cleavage of huntingtin by apopain,

210 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. Neuron Matrix Metalloproteinases Modify Huntingtin

a proapoptotic cysteine protease, is modulated by the polyglutamine tract. non-redundant proteolytic functions of the presenilin homologues SPPL2b Nat. Genet. 13, 442–449. and SPPL3. J. Biol. Chem. 280, 39515–39523. Gourfinkel-An, I., Cancel, G., Trottier, Y., Devys, D., Tora, L., Lutz, Y., Imbert, Landles, C., Sathasivam, K., Weiss, A., Woodman, B., Moffitt, H., Finkbeiner, G., Saudou, F., Stevanin, G., Agid, Y., et al. (1997). Differential distribution of S., Sun, B., Gafni, J., Ellerby, L.M., Trottier, Y., et al. (2010). Proteolysis of the normal and mutated forms of huntingtin in the human brain. Ann. Neurol. mutant huntingtin produces an exon 1 fragment that accumulates as an aggre- 42, 712–719. gated protein in neuronal nuclei in Huntington disease. J. Biol. Chem. 285, Graham, R.K., Deng, Y., Slow, E.J., Haigh, B., Bissada, N., Lu, G., Pearson, J., 8808–8823. Shehadeh, J., Bertram, L., Murphy, Z., et al. (2006). Cleavage at the caspase-6 Lee, H., Park, J.W., Kim, S.P., Lo, E.H., and Lee, S.R. (2009). Doxycycline site is required for neuronal dysfunction and degeneration due to mutant inhibits matrix metalloproteinase-9 and laminin degradation after transient huntingtin. Cell 125, 1179–1191. global cerebral ischemia. Neurobiol. Dis. 34, 189–198. Gu, Z., Cui, J., Brown, S., Fridman, R., Mobashery, S., Strongin, A.Y., and Lineberry, N., Su, L., Soares, L., and Fathman, C.G. (2008). The single subunit Lipton, S.A. (2005). A highly specific inhibitor of matrix metalloproteinase-9 transmembrane E3 ligase gene related to anergy in lymphocytes (GRAIL) rescues laminin from proteolysis and neurons from apoptosis in transient focal captures and then ubiquitinates transmembrane proteins across the cell cerebral ischemia. J. Neurosci. 25, 6401–6408. membrane. J. Biol. Chem. 283, 28497–28505. Hackam, A.S., Singaraja, R., Wellington, C.L., Metzler, M., McCutcheon, K., Llano, E., Penda´ s, A.M., Aza-Blanc, P., Kornberg, T.B., and Lo´ pez-Otıń, C. Zhang, T., Kalchman, M., and Hayden, M.R. (1998). The influence of huntingtin (2000). Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential protein size on nuclear localization and cellular toxicity. J. Cell Biol. 141, role in extracellular matrix remodeling during neural development. J. Biol. 1097–1105. Chem. 275, 35978–35985. Hedreen, J.C., Peyser, C.E., Folstein, S.E., and Ross, C.A. (1991). Neuronal Lunkes, A., Lindenberg, K.S., Ben-Haıëm, L., Weber, C., Devys, D., Landwehr- loss in layers V and VI of cerebral cortex in Huntington’s disease. Neurosci. meyer, G.B., Mandel, J.L., and Trottier, Y. (2002). Proteases acting on mutant Lett. 133, 257–261. huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol. Cell 10, 259–269. Hermel, E., Gafni, J., Propp, S.S., Leavitt, B.R., Wellington, C.L., Young, J.E., Hackam, A.S., Logvinova, A.V., Peel, A.L., Chen, S.F., et al. (2004). Specific Luo, D., Mari, B., Stoll, I., and Anglard, P. (2002). Alternative splicing and caspase interactions and amplification are involved in selective neuronal promoter usage generates an intracellular stromelysin 3 isoform directly trans- 277 vulnerability in Huntington’s disease. Cell Death Differ. 11, 424–438. lated as an active matrix metalloproteinase. J. Biol. Chem. , 25527–25536. Hornebeck, W., and Lafuma, C. (1991). Matrix metalloproteinase (MMP). C. R. Malemud, C.J. (2006). Matrix metalloproteinases (MMPs) in health and 11 Seances Soc. Biol. Fil. 185, 127–134. disease: an overview. Front. Biosci. , 1696–1701. The Huntington’s Disease Collaborative Research Group. (1993). A novel gene Mao, Z., Choo, Y.S., and Lesort, M. (2006). Cystamine and cysteamine prevent containing a trinucleotide repeat that is expanded and unstable on Hunting- 3-NP-induced mitochondrial depolarization of Huntington’s disease knock-in 23 ton’s disease chromosomes. Cell 72, 971–983. striatal cells. Eur. J. Neurosci. , 1701–1710. Kaltenbach, L.S., Romero, E., Becklin, R.R., Chettier, R., Bell, R., Phansalkar, Martindale, D., Hackam, A., Wieczorek, A., Ellerby, L., Wellington, C., A., Strand, A., Torcassi, C., Savage, J., Hurlburt, A., et al. (2007). Huntingtin McCutcheon, K., Singaraja, R., Kazemi-Esfarjani, P., Devon, R., Kim, S.U., interacting proteins are genetic modifiers of neurodegeneration. PLoS Genet. et al. (1998). Length of huntingtin and its polyglutamine tract influences local- 18 3, e82. 10.1371/journal.pgen.0030082. ization and frequency of intracellular aggregates. Nat. Genet. , 150–154. Kegel, K.B., Sapp, E., Alexander, J., Valencia, A., Reeves, P., Li, X., Masso, N., Mende-Mueller, L.M., Toneff, T., Hwang, S.R., Chesselet, M.F., and Hook, V.Y. Sobin, L., Aronin, N., and Difiglia, M. (2009a). Polyglutamine expansion in (2001). Tissue-specific proteolysis of Huntingtin (htt) in human brain: evidence huntingtin alters its interaction with phospholipids. J. Neurochem. 110, of enhanced levels of N- and C-terminal htt fragments in Huntington’s disease 21 1585–1597. striatum. J. Neurosci. , 1830–1837. Milakovic, T., and Johnson, G.V. (2005). Mitochondrial respiration and ATP Kegel, K.B., Schewkunow, V., Sapp, E., Masso, N., Wanker, E.E., DiFiglia, M., production are significantly impaired in striatal cells expressing mutant hun- and Goldmann, W.H. (2009b). Polyglutamine expansion in huntingtin increases tingtin. J. Biol. Chem. 280, 30773–30782. its insertion into lipid bilayers. Biochem. Biophys. Res. Commun. 387, 472–475. Mookerjee, S., Papanikolaou, T., Guyenet, S.J., Sampath, V., Lin, A., Vitelli, C., DeGiacomo, F., Sopher, B.L., Chen, S.F., La Spada, A.R., and Ellerby, L.M. Kim, M., Lee, H.S., LaForet, G., McIntyre, C., Martin, E.J., Chang, P., Kim, (2009). Posttranslational modification of ataxin-7 at lysine 257 prevents T.W., Williams, M., Reddy, P.H., Tagle, D., et al. (1999). Mutant huntingtin autophagy-mediated turnover of an N-terminal caspase-7 cleavage fragment. expression in clonal striatal cells: dissociation of inclusion formation and J. Neurosci. 29, 15134–15144. neuronal survival by caspase inhibition. J. Neurosci. 19, 964–973. Oliveira, J.M., Chen, S., Almeida, S., Riley, R., Gonçalves, J., Oliveira, C.R., Kim, Y.J., Yi, Y., Sapp, E., Wang, Y., Cuiffo, B., Kegel, K.B., Qin, Z.H., Aronin, Hayden, M.R., Nicholls, D.G., Ellerby, L.M., and Rego, A.C. (2006). Mitochon- N., and DiFiglia, M. (2001). Caspase 3-cleaved N-terminal fragments of wild- drial-dependent Ca2+ handling in Huntington’s disease striatal cells: effect of type and mutant huntingtin are present in normal and Huntington’s disease histone deacetylase inhibitors. J. Neurosci. 26, 11174–11186. brains, associate with membranes, and undergo calpain-dependent proteolysis. Proc. Natl. Acad. Sci. USA 98, 12784–12789. Page-McCaw, A. (2008). Remodeling the model organism: matrix metalloproteinase functions in invertebrates. Semin. Cell Dev. Biol. 19, 14–23. Kim, Y.J., Sapp, E., Cuiffo, B.G., Sobin, L., Yoder, J., Kegel, K.B., Qin, Z.H., Detloff, P., Aronin, N., and DiFiglia, M. (2006). Lysosomal proteases are Page-McCaw, A., Serano, J., Sante´ , J.M., and Rubin, G.M. (2003). Drosophila involved in generation of N-terminal huntingtin fragments. Neurobiol. Dis. 22, matrix metalloproteinases are required for tissue remodeling, but not embry- 4 346–356. onic development. Dev. Cell , 95–106. Konttinen, Y.T., Ainola, M., Valleala, H., Ma, J., Ida, H., Mandelin, J., Kinne, Pei, D., and Weiss, S.J. (1995). Furin-dependent intracellular activation of the 375 R.W., Santavirta, S., Sorsa, T., Lo´ pez-Otıń, C., and Takagi, M. (1999). Analysis human stromelysin-3 zymogen. Nature , 244–247. of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial Pei, D., and Weiss, S.J. (1996). Transmembrane-deletion mutants of the membrane: different profiles in trauma and rheumatoid arthritis. Ann. Rheum. membrane-type matrix metalloproteinase-1 process progelatinase A and Dis. 58, 691–697. express intrinsic matrix-degrading activity. J. Biol. Chem. 271, 9135–9140. Krawitz, P., Haffner, C., Fluhrer, R., Steiner, H., Schmid, B., and Haass, C. Ratovitski, T., Nakamura, M., D’Ambola, J., Chighladze, E., Liang, Y., Wang, (2005). Differential localization and identification of a critical aspartate suggest W., Graham, R., Hayden, M.R., Borchelt, D.R., Hirschhorn, R.R., and Ross,

Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc. 211 Neuron Matrix Metalloproteinases Modify Huntingtin

C.A. (2007). N-terminal proteolysis of full-length mutant huntingtin in an induc- huntingtin in mouse and monkey models implicated as a pathogenic mecha- ible PC12 cell model of Huntington’s disease. Cell Cycle 6, 2970–2981. nism in Huntington’s disease. Hum. Mol. Genet. 17, 2738–2751. Ratovitski, T., Gucek, M., Jiang, H., Chighladze, E., Waldron, E., D’Ambola, J., Wei, S., Xie, Z., Filenova, E., and Brew, K. (2003). Drosophila TIMP is a potent Hou, Z., Liang, Y., Poirier, M.A., Hirschhorn, R.R., et al. (2009). Mutant hunting- inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3. tin N-terminal fragments of specific size mediate aggregation and toxicity in Biochemistry 42, 12200–12207. neuronal cells. J. Biol. Chem. 284, 10855–10867. Ronco, P., Lelongt, B., Piedagnel, R., and Chatziantoniou, C. (2007). Matrix Wellington, C.L., Ellerby, L.M., Hackam, A.S., Margolis, R.L., Trifiro, M.A., metalloproteinases in kidney disease progression and repair: a case of flipping Singaraja, R., McCutcheon, K., Salvesen, G.S., Propp, S.S., Bromm, M., the coin. Semin. Nephrol. 27, 352–362. et al. (1998). Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglut- Silvestroni, A., Faull, R.L., Strand, A.D., and Mo¨ ller, T. (2009). Distinct neuroin- amine tract. J. Biol. Chem. 273, 9158–9167. flammatory profile in post-mortem human Huntington’s disease. Neuroreport 20, 1098–1103. Wellington, C.L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Catta- Tanaka, Y., Igarashi, S., Nakamura, M., Gafni, J., Torcassi, C., Schilling, G., neo, E., Hackam, A., Sharp, A., Thornberry, N., et al. (2000). Inhibiting caspase Crippen, D., Wood, J.D., Sawa, A., Jenkins, N.A., et al. (2006). Progressive cleavage of huntingtin reduces toxicity and aggregate formation in neuronal phenotype and nuclear accumulation of an amino-terminal cleavage fragment and nonneuronal cells. J. Biol. Chem. 275, 19831–19838. in a transgenic mouse model with inducible expression of full-length mutant Wellington, C.L., Ellerby, L.M., Gutekunst, C.A., Rogers, D., Warby, S., huntingtin. Neurobiol. Dis. 21, 381–391. Graham, R.K., Loubser, O., van Raamsdonk, J., Singaraja, R., Yang, Y.Z., Trettel, F., Rigamonti, D., Hilditch-Maguire, P., Wheeler, V.C., Sharp, A.H., et al. (2002). Caspase cleavage of mutant huntingtin precedes neurodegener- Persichetti, F., Cattaneo, E., and MacDonald, M.E. (2000). Dominant pheno- ation in Huntington’s disease. J. Neurosci. 22, 7862–7872. types produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 9, 2799–2809. Xue, M., Fan, Y., Liu, S., Zygun, D.A., Demchuk, A., and Yong, V.W. (2009). Vergote, D., Butler, G.S., Ooms, M., Cox, J.H., Silva, C., Hollenberg, M.D., Contributions of multiple proteases to neurotoxicity in a mouse model of intra- 132 Jhamandas, J.H., Overall, C.M., and Power, C. (2006). Proteolytic processing cerebral haemorrhage. Brain , 26–36. of SDF-1alpha reveals a change in receptor specificity mediating HIV-associ- Yanai, A., Huang, K., Kang, R., Singaraja, R.R., Arstikaitis, P., Gan, L., Orban, ated neurodegeneration. Proc. Natl. Acad. Sci. USA 103, 19182–19187. P.C., Mullard, A., Cowan, C.M., Raymond, L.A., et al. (2006). Palmitoylation of Wang, C.E., Tydlacka, S., Orr, A.L., Yang, S.H., Graham, R.K., Hayden, M.R., huntingtin by HIP14 is essential for its trafficking and function. Nat. Neurosci. 9, Li, S., Chan, A.W., and Li, X.J. (2008). Accumulation of N-terminal mutant 824–831.

212 Neuron 67, 199–212, July 29, 2010 ª2010 Elsevier Inc.